Inhibition of NF-kappa B by S-nitrosylation.

It is not clear if redox regulation of transcription is the consequence of direct redox-related modifications of transcription factors, or if it occurs at some other redox-sensitive step. One obstacle has been the inability to demonstrate redox-related modifications of transcription factors in vivo. The redox-sensitive transcriptional activator NF-kappaB (p50-p65) is a case in point. Its activity in vitro can be inhibited by S-nitrosylation of a critical thiol in the DNA-interacting p50 subunit, but modulation of NF-kappaB activity by nitric oxide synthase (NOS) has been attributed to other mechanisms. Herein we show that cellular NF-kappaB activity is in fact regulated by S-nitrosylation. We observed that both S-nitrosocysteine and cytokine-activated NOS2 inhibited NF-kappaB in human respiratory cells or murine macrophages. This inhibition was reversed by addition of the denitrosylating agent dithiothreitol to cellular extracts, whereas NO bioactivity did not affect the TNFalpha-induced degradation of IkappaBalpha or the nuclear translocation of p65. Recapitulation of these conditions in vitro resulted in S-nitrosylation of recombinant p50, thereby inhibiting its binding to DNA, and this effect was reversed by dithiothreitol. Further, an increase in S-nitrosylated p50 was detected in cells, and the level was modulated by TNFalpha. Taken together, these data suggest that S-nitrosylation of p50 is a physiological mechanism of NF-kappaB regulation.

Nitrosation and oxidation in the regulation of gene expression.

A growing body of evidence suggests that the cellular response to oxidative and nitrosative stress is primarily regulated at the level of transcription. Posttranslational modification of transcription factors may provide a mechanism by which cells sense these redox changes. In bacteria, for example, OxyR senses redox-related changes via oxidation or nitrosylation of a free thiol in the DNA binding region. This mode of regulation may serve as a paradigm for redox-sensing by eukaryotic transcription factors as most-including NF-kappaB, AP-1, and p53-contain reactive thiols in their DNA binding regions, the modification of which alters binding in vitro. Several of these transcription factors have been found to be sensitive to both reactive oxygen species and nitric oxide-related species in vivo. It remains entirely unclear, however, if oxidation or nitrosylation of eukaryotic transcription factors is an important mode of regulation, or whether transcriptional activating pathways are principally controlled at other redox-sensitive levels.-Marshall, H. E., Merchant, K., Stamler, J. S. Nitrosation and oxidation in the regulation of gene expression.

Thymus-dependent, anti-CD4-induced tolerance to rat cardiac allografts.

BACKGROUND: Treatment with anti-CD4 monoclonal antibodies (mAbs) leads to induction of transplant tolerance in rodent models, but the cellular mechanisms responsible are poorly defined. In this study, we used a rat model of cardiac transplantation to examine the contribution of the thymus gland to anti-CD4 mAb-induced tolerance. METHODS: Pretransplant administration of OX38 mAb partially depletes peripheral CD4 T cells and induces tolerance to fully allogeneic Lewis (RT1l) heterotopic cardiac allografts in DA (RT1a) recipients. Using this experimental model, the contribution of the adult thymus gland and of recent thymic emigrants to tolerance induction was assessed, and the cellular and humoral alloimmune responses accompanying tolerance defined. RESULTS: OX38 mAb selectively depleted mature CD4 T cells but spared CD4 T cells that had recently emerged from the thymus. Pretransplant thymectomy abrogated tolerance induction, but the data suggested a role for recent thymic emigrants rather than for the thymus gland per se. Both nonrejecting cardiac allografts in OX38-treated recipients and rejecting grafts in control animals were infiltrated to a similar extent by mononuclear cells, including activated T cells. Intragraft mRNA transcripts for interleukin (IL)-2, interferon-gamma, IL-4, IL-10, and IL-13 were similar in non-rejecting and rejecting allografts although, with the exception of IL-2, there was a trend towards reduced cytokine transcripts in tolerant grafts. CD4 T cells from long-term tolerant recipients proliferated normally to donor alloantigen in vitro, and produced IL-2, interferon-gamma, and IL-4 in amounts comparable to normal CD4 T cells. Tolerant recipients also developed a strong alloantibody response comprising both IgG1 (Th2-dependent) and IgG2b (Th1-dependent) subclasses. CONCLUSIONS: The results of this study suggest that the thymus, through the production of recent thymic emigrants, plays an important role in facilitating the induction of transplant tolerance after anti-CD4 mAb. Tolerant animals displayed strong cell-mediated and humoral alloimmune responses with no evidence of selective deviation from a Th1 to a Th2-like cytokine pattern.

Alloantibody and intragraft cellular response to MHC class I-disparate kidney allografts in recipients tolerized by donor-specific transfusion and cyclosporine.

Congenic PVG.RT1u rats rapidly reject Aa class I-disparate kidney allografts from recombinant PVG R8 donors and we recently demonstrated that anti-class I MHC alloantibody plays a critical role in effecting acute rejection in this experimental model. In this article, we show that PVG.RT1u recipients can be rendered permanently and specifically tolerant to R8 kidney allografts by administration of four weekly donor-specific transfusions (DST) combined with a 7-day course of cyclosporine given with the first DST. Tolerance induction correlated with abrogation of a cytotoxic alloantibody response by thymus-independent, i.e., peripheral mechanisms; IgM and all IgG subclasses of anti-class I alloantibody were abolished. In contrast, nonrejecting kidney allografts in tolerant rats and rejecting grafts from unmodified recipients were similarly infiltrated by mononuclear cells, and intragraft transcripts for interleukin (IL)-2, interferon-gamma, and IL-13 were readily detected by reverse transcriptase polymerase chain reaction with no apparent quantitative difference between the two groups. Messenger RNA for IL-4 and IL-10 was present in rejecting grafts but barely detectable in grafts from tolerant animals. These results suggest that tolerance induction by DST and cyclosporine is, in this experimental model, associated with a selective impairment in humoral alloimmunity.

CD4+ T cell-mediated rejection of major histocompatibility complex class I-disparate grafts: a role for alloantibody.

Experimental studies of the T cell requirement for rejection of class I major histocompatibility complex (MHC)-disparate grafts have generated controversy over both the autonomy of CD8+ T cells and the mechanism whereby CD4+ T cells are able to independently mediate rejection. In this study of rejection of RT1Aa class I MHC-disparate rat cardiac and skin allografts by high-responder PVG RT1u recipients, we show that elimination of CD8+ T cells [by anti-CD8 monoclonal antibody (mAb) administration in vivo] fails to prolong graft survival, whereas partial depletion of CD4+ T cells (by anti-CD4 mAb treatment) markedly delays rejection of class I-disparate heart grafts, and marginally prolongs survival of skin grafts. Anti-CD4-treated PVG-RT1u athymic nude rats reconstituted with CD8+ T cells failed to reject class I-disparate skin grafts for several weeks and eventual rejection correlated with re-emergence of a small number of donor derived CD4+ T cells. Conversely, anti-CD8-treated nude rats reconstituted with CD4+ T cells alone rapidly rejected class I-disparate skin grafts. Passive transfer of anti-class I immune serum to anti-CD4-treated euthymic recipients promptly restored their ability to specifically reject a class I-disparate heart graft. Similarly, passive transfer of immune serum to PVG-RT1u nude rats bearing skin allografts caused destruction of class I-disparate but not third-party grafts. These results demonstrate that CD4+ T cells are both necessary and sufficient to cause rejection of class I-disparate heart and skin grafts in this model and that CD4+ T cell-dependent alloantibody plays a decisive role in effecting rejection.

Cytomegalovirus endomyocarditis in a transplanted heart. A case report with in situ hybridization.

A 64-year-old man underwent cardiac transplantation for long-standing severe dilated cardiomyopathy. Postoperative complications included primary cytomegalovirus (CMV) infection with several episodes of moderate acute rejection and severe pneumonia. Six months after transplantation, an endomyocardial biopsy specimen revealed focal necrotizing myocarditis with intranuclear inclusions consistent with CMV. The patient subsequently developed fulminant pneumonia and died 7 months after transplantation. Postmortem examination revealed that the cause of death was acute necrotizing bronchopneumonia due to Staphylococcus aureus, with underlying CMV pneumonitis. The transplanted heart had left ventricular hypertrophy with multiple organizing myocardial infarcts, moderate coronary atherosclerosis, and organizing thrombi of the left atrium. Characteristic inclusions of CMV were identified, predominantly within endothelial cells, in the left coronary artery, left ventricular endocardium, and myocardium. With in situ hybridization, the presence of CMV was verified in the inclusions, as well as in many fibroblasts without inclusions. In situ hybridization is warranted in myocardial biopsy specimens when suspicious inclusions or infiltrates are present, to confirm CMV infection, so that appropriate therapy can be initiated.

Administrative physiatry.

Results of a mail survey of physiatrists and residents in physical medicine and rehabilitation indicate that physiatrists need administrative/managerial skills in their practices. For physiatrists to initiate new programs, they need skills in program planning and development, resource management, and similar areas. Experience gained as chief resident appears to have a positive bearing on gaining administrative/managerial skills. Similarly, experience in dealing with multidisciplinary treatment teams also seems to help. However, survey results indicate that formal training to acquire administrative/managerial skills or to lead multidisciplinary treatment teams is not offered in many residency programs. Physical medicine and rehabilitation residency curricula require that residents learn management techniques consistent with their team leadership roles. We suggest several methods of offering formal training to residents and to physiatrists in practice, via continuing education.

FcR blocking activity in serum of actively enhanced rat renal allograft recipients due to IgG anti-class II MHC alloantibody.

In some rat strain combinations, pre-operative donor-specific blood transfusion produces long-term renal allograft survival, although the underlying mechanisms are unclear. This study has examined whether Fc receptor (FcR)-blocking activity could be detected in the serum of unmodified PVG strain recipients bearing a rejecting renal allograft and in recipients bearing an actively enhanced graft following pre-operative blood transfusion. Serum harvested on Day 5 from actively enhanced PVG recipients of DA rat renal allografts was shown to specifically inhibit erythrocyte-antibody (EA) rosette formation with donor strain, but not third-party, splenocytes, while the levels of EA rosette inhibition (EAI) in Day 5 serum from rejecting rats remained markedly lower. This FcR-blocking activity was present in enhanced serum fractions, prepared by discontinuous density gradient centrifugation, which corresponded to the 7 S peak. Purified IgG prepared from enhanced serum was also found to inhibit EA rosette formation with donor splenocytes, and absorption of the IgG preparations with donor strain erythrocytes failed to abrogate EA rosette inhibition. Further experiments, in which absorbed IgG from enhanced animals was tested for FcR blocking activity against splenocytes of defined major histocompatability complex (MHC) subregion specificities, established that FcR-blocking activity was mediated by IgG alloantibodies directed against donor MHC class II antigens. Whether the presence of such antibodies early after transplantation contributes to the beneficial effect of blood transfusion on graft survival remains to be determined.

The stathmokinetic and morphological response of the hamster respiratory epithelium to intralaryngeal instillations of saline and ferric oxide in saline.

Ferric oxide (Fe2O3) and saline, substances which enhance lung tumor development when intratracheally instilled with certain carcinogens, were studied for their effects on respiratory epithelial cell proliferation in Syrian golden hamsters. Groups of hamsters were given intralaryngeal instillations of 0.2 ml saline or 3 mg Fe2O3 in 0.2 ml saline (Fe2O3-saline) once a week for 5, 10, or 15 weeks; an additional group of hamsters received a single Fe2O3-saline instillation. Epithelial mitotic rates (MR, 6 hr colchicine blockade) for the midtrachea, left intrapulmonary bronchus, and left lung bronchioles were determined 32 hr after the last instillation for each group. No change in the MR was seen at any airway level for any of the saline-treated groups. There was also no change in the MR of the trachea or bronchus for any of the groups treated with Fe2O3-saline. The MR of the bronchioles was increased after 5 instillations of Fe2O3-saline. Fe2O3-saline instillations also caused bronchioloalveolar hyperplasia (BAH) to develop by 5 instillations, but after 10 and 15 treatments BAH was less prominent. These data indicate that intralaryngeal Fe2O3-saline instillations result in focal cell injury followed by proliferation in the distal airways, but multiple long-term treatments produce an adaptive response of the respiratory epithelium.

Abrupt memory loss associated with thalamic tumor.

We report a case in which glioma with apparent major involvement of the right thalamus was manifested initially solely by abrupt onset of severe impairment of both recent and moderately remote memory. Distracting stimuli plays a role in prevention of consolidation of memory.

The removal of an esophageal papilloma by endoscopic means. A case report and brief review of the literature.

The case of a 77-year old white male on anticoagulant therapy who developed sudden onset of gastrointestinal bleeding is presented. A benign esophageal papilloma was a fortuitous finding. The papilloma was removed in toto by fiberoptic endoscopic polypectomy. A brief review of this rare lesion is presented and the importance of a careful endoscopic examination as well as the therapeutic/curative potentialities of the procedure are stressed.